Since a mesenchymal stem cell recently has been isolated successfully from human, a clinical application of the mesenchymal stem cell has been brought to a focus. Particularly, the clinical application of the mesenchymal stem cell is used as cell donor for a cell replacement therapy. The cell replacement therapy is presented for effectively treating cell deficiency caused by neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease known by incurable disease, ischemic and hemorrhagic stroke, traumatic disease and spinal cord injury, which are caused by destruction and permanent functional disorder of cells consisting of tissues.
However, cell replacement therapy has a limitation in practical application. That is, a conventional method of implanting cells differentiated fully into donor's tissues to patients is difficult to obtain sufficient amount of the cells to give the patients.
In order to solve the above-mentioned problems, differentiating the mesenchymal stem cell into a tissue-specific cell or inducing differentiation after isolating and proliferating the tissue-specific stem cell may be used as the cell replacement therapy.
Implanting a neural cell differentiated from a mesenchymal stem cell in one human into another human, however, is practically difficult, and creates immunological reactions in case of implanting the neural cell between male and female cell donors.
Until now, differentiations of a mesenchymal stem cell of a rat into a hematopoietic cell, a myocardial cell, an islet of Langerhans and a neural cell are proved in culture flask. Studies such as a study on implanting an oligodendrocyte into a rat and being increased to create myelin after inducing to differentiate into the oligodendrocyte as a cell creating myelin from the mesenchymal stem cell (Brustle et al., Science 285: 754-756), a study on implanting an insulin secreting cell, differented from the mesenchymal stem cell, into a diabetic rat and regulating blood sugar lever (Soria et al., Diabetes 49:11157-162, 2000), a study on implanting a neural cell that is differentiated from the mesenchymal stem cell, into a spinal cord injured rat and confirming to improve motor disturbance spinal cord injured (McDonald et al., Nat. Med. 5(12):1410-1412, 1999), etc., represent a method of implanting of the cells that are differentiated from the mesenchymal stem cell, for effectively treating diseases due to deficiency of cell.
However, isolation of the mesenchymal stem cell has been accomplished just recently and differentiation of the mesenchymal stem cell into the other cells except the neural cell in culture flask has been yet reported, such that the clinical application of the tissue-specific cell differentiated from the mesenchymal stem cell for the cell replacement therapy is possible, but is not practical.
Although a method of differentiating a cell from the mesenchymal stem cell as for the cell replacement therapy is thought as the most effective method until now, a risk of other cells mixed due to low efficiency of differentiating into the tissue-specific cell from the mesenchymal stem cell except immunological reactions in case of implanting into a patient still exists, such that studies on delicate differentiation are required for safe clinical application.
A method of using the tissue-specific stem cell for the cell replacement therapy also has a problem such as differentiating into undesired cells due to modifying of differentiation potential when the tissue-specific stem cell is cultured for a long time, decreasing of proliferation rate of a cell. Moreover, a neural cell for treating neurodegenerative disease such as Parkinson's disease is required to be implanted. Considering that the neural cell is obtained mainly by differentiating and proliferating a neural stem cell from fetal brain since the neural cell is difficult to be obtained directly from patients, implanting a neural cell is a disadvantage. About two fetal brains are required to treat one patient in such a case of above. Implanting the neural cell has problems such as insufficient supply of the neural cell and unethical act, differentiation of the neural stem cell into an astrocyte than the neural cell, and generation of immunological reaction.
Accordingly, if a method of treatment using the neural cell differentiated from the mesenchymal stem cell is possible, problems such as difficulty in obtaining sufficient cells and generating immunological reaction may be solved. Whether the mesenchymal stem cell as a mesoderm may be differentiated into the neural cell is still in question, but trans-differentiation was recently reported and the possibility of differentiation of the mesenchymal stem cell is increased.
Formerly, a hepatocyte is known to be differentiated into only specific class of a tissue cell. It was reported that the mesenchymal stem cell formed in vitro colonies in a medium comprising growth factors such as basic fibroblast growth factor, transforming growth factor, epithermal growth factor etc. (Kuznetsov et al., Br. J. Haematol. 97:561, 1997; Van den Bos C et al., Human Cell 10:45, 1997). In addition, about a third of the firstly anchored cells with multi-differentiative potential were differentiated into desmoplastic cells such as osteoblast, chondroblast, adipocyte etc. (Pttenger M F et al., Science 279:1528, 1998), and a bone marrow was resource of a myogenic precursor cell for forming new muscles (Ferrari G et al., Science 279:1528, 1998).
However, according to recent consecutive studies, it was reported that the mesenchymal stem cell might be differentiated into not only desmoplastic cells, but also into neural cells. For example, it was reported that the mesenchymal stem cell was differentiated into the neural cell and the astrocyte by culturing in medium comprising retinoic acid and BDNF (brain-derived neurotrophic factor) (Sanchez-Ramos et al., Exp. Neurology 164:247-256, 2000) and into the neural cell by culturing in medium comprising antioxidative substance such as mercaptoethanol and DMSO (dimethyl sulfoxide) (Dale Woodbury et al., J. Neuro. Res. 61:364-370, 2000).
When a chemical reagent such as DMSO is used to induce differentiation, however, toxicity of DMSO may alter the mesenchymal stem cell such that a safer method is required.
The inventor of the present invention studied a safe and effective method of differentiating of the mesenchymal stem cell into the neural cell, and applied for a patent that was “method of differentiating the mesenchymal stem cell into the neural cell” at Korean patent number 2001-21064. The patent is related to a method of differentiating and proliferating the mesenchymal stem cell into the neural cell by culturing in medium comprising an epithermal growth factor and a hepatocyte growth factor.
The method of differentiating and proliferating of the mesenchymal stem cell into the neural cell has problems not only with low rate but also about 80% efficiency of differentiation. Further, culturing for four weeks is required due to low differentiation rate when a cell during differentiating and proliferating is cultured such that morphological alteration occurs. Thus, problems such as contamination and a large consumption of reagents, equipments and time, are generated.
The method in Korean patent number 2001-21064 is proposing the possibility that the neural cell that is differentiated from the mesenchymal stem cell is useful for treating neurodegenerative disease such as Parkinson's disease, Alzheimer, Pick's disease, Huntington's disease, amyotrophic lateral sclerosis, and ischemic and hemorrhagic brain disease, however, in vitro test, animal test or clinical test has yet performed and thus pharmacological effect of the neural cell also has yet confirmed.
To solve the problems, the inventor completed the present invention by studying on a method of improving differentiation rate and efficiency, and a test of pharmacological effect.